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Article info Received: 28 September 2017; Accepted: 14 March 2018; Published: 21 June 2018. ...... Inter-Society Color Council – National Bureau of Standards color name charts .... Plant Pathology 57: 33–44. Spiers A, Hopcroft D. 1994.
Persoonia 41, 2018: 142 –174 www.ingentaconnect.com/content/nhn/pimj

RESEARCH ARTICLE

ISSN (Online) 1878-9080 https://doi.org/10.3767/persoonia.2018.41.08

Unravelling species boundaries in the Aspergillus viridinutans complex (section Fumigati): opportunistic human and animal pathogens capable of interspecific hybridization V. Hubka1,2,3*, V. Barrs 4#, Z. Dudová1,3#, F. Sklenář 1,2#, A. Kubátová1, T. Matsuzawa 5, T. Yaguchi 6, Y. Horie 6, A. Nováková 2, J.C. Frisvad 7, J.J. Talbot 4, M. Kolařík 2

Key words Aspergillus felis Aspergillus fumigatus invasive aspergillosis mating-type genes multispecies coalescence model Neosartorya udagawae scanning electron microscopy soil fungi

Abstract   Although Aspergillus fumigatus is the major agent of invasive aspergillosis, an increasing number of infections are caused by its cryptic species, especially A. lentulus and the A. viridinutans species complex (AVSC). Their identification is clinically relevant because of antifungal drug resistance and refractory infections. Species boundaries in the AVSC are unresolved since most species have uniform morphology and produce interspecific hybrids in vitro. Clinical and environmental strains from six continents (n = 110) were characterized by DNA sequencing of four to six loci. Biological compatibilities were tested within and between major phylogenetic clades, and ascospore morphology was characterised. Species delimitation methods based on the multispecies coalescent model (MSC) supported recognition of ten species including one new species. Four species are confirmed opportunistic pathogens; A. udagawae followed by A. felis and A. pseudoviridinutans are known from opportunistic human infections, while A. felis followed by A. udagawae and A. wyomingensis are agents of feline sino-orbital aspergillosis. Recently described human-pathogenic species A. parafelis and A. pseudofelis are synonymized with A. felis and an epitype is designated for A. udagawae. Intraspecific mating assay showed that only a few of the heterothallic species can readily generate sexual morphs in vitro. Interspecific mating assays revealed that five different species combinations were biologically compatible. Hybrid ascospores had atypical surface ornamentation and significantly different dimensions compared to parental species. This suggests that species limits in the AVSC are maintained by both pre- and post-zygotic barriers and these species display a great potential for rapid adaptation and modulation of virulence. This study highlights that a sufficient number of strains representing genetic diversity within a species is essential for meaningful species boundaries delimitation in cryptic species complexes. MSC-based delimitation methods are robust and suitable tools for evaluation of boundaries between these species. Article info   Received: 28 September 2017; Accepted: 14 March 2018; Published: 21 June 2018.

INTRODUCTION Aspergillus is a speciose genus with almost 400 species classi­ fied into six subgenera and approximately 25 sections (Samson et al. 2014, Jurjević et al. 2015, Hubka et al. 2016a, 2017, Chen et al. 2016a, b, 2017, Kocsubé et al. 2016, Sklenář et al. 2017, Tanney et al. 2017). The species are widely distributed in nature and have a significant economic impact in human and animal health (causative agents of aspergillosis; allergies and respiratory problems associated with presence of fungi in the indoor environment), the food industry (source of enzymes and organic acids for fermentation, food and feed spoilage, production of Department of Botany, Faculty of Science, Charles University, Benátská 2, 128 01 Prague 2, Czech Republic. 2 Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 142 20 Prague 4, Czech Republic. 3 First Faculty of Medicine, Charles University, Kateřinská 32, 121 08 Prague 2, Czech Republic. 4 Sydney School of Veterinary Science, Faculty of Science, and Marie Bashir Institute of Infectious Diseases & Biosecurity, University of Sydney, Camper­ down, NSW, Australia. 5 University of Nagasaki, 1-1-1 Manabino, Nagayo-cho, Nishi-Sonogi-gun, Nagasaki 851-2195, Japan. 6 Medical Mycology Research Center, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8673, Japan. 7 Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. * corresponding author e-mail: [email protected]. # These co-authors contributed equally to this work. 1

hazardous mycotoxins), biotechnology and pharmacology (production of bioactive substances, heterologous proteins) (Pitt & Hocking 2009, Meyer et al. 2011, Frisvad & Larsen 2015b, Sugui et al. 2015, Gautier et al. 2016). Aspergillus sect. Fumigati includes approximately 60 species occurring predominantly in soil (Hubka et al. 2017). Many are of considerable medical importance as they cause human and animal infections (Balajee et al. 2005b, 2009, Katz et al. 2005, Yaguchi et al. 2007, Hubka et al. 2012, Talbot & Barrs 2018). Aspergillus fumigatus is usually reported as both the most common member of the section in soil worldwide and the most common cause of aspergillosis (Klich 2002, Domsch et al. 2007, Mayr & Lass-Flörl 2011). A series of recent studies highlighted the high prevalence (11–19 %) of so-called cryptic Aspergillus species in clinical samples (Balajee et al. 2009, Alastruey-Izquierdo et al. 2013, Negri et al. 2014, Sabino et al. 2014). Their identification is clinically relevant since many demonstrate drug resistance to commonly used antifungals, thus their recognition influences therapeutic management. Reliable identification of clinical isolates to the species level and susceptibility testing by reference methods is thus warranted (Lyskova et al. 2018). Many of these less common pathogens belong to sect. Fumigati and the highest numbers of infections are attributed to A. lentulus, A. thermomutatus (syn. Neosartorya pseudofischeri ) and species from A. viridinutans species complex (AVSC) (Balajee et al. 2005a, 2006, Sugui et al. 2010, 2014, Barrs et al. 2013, Talbot & Barrs 2018).

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homothallic homothallic homothallic homothallic MAT1-2-1 (KC797620) MAT1-2-1 (KJ858505) MAT1-2-1 (KJ858507) MAT1-1-1 (KJ858506) MAT1-2-1 MAT1-1-1 (HF937392) MAT1-1-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 (LT796767) MAT1-2-1 MAT1-2-1 MAT1-2-1 (LT796766) ND MAT1-1-1 (LT796760) MAT1-1-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 (KC797622) MAT1-1-1 (KC797627) MAT1-2-1 (KC797625) ND ND MAT1-1-1 (KC797629) MAT1-2-1 MAT1-2-1 (KC797621) MAT1-2-1 MAT1-2-1 MAT1-1-1 (KC797632)

Brazil, São Paulo State, Botucatú, soil, 1993 Brazil, Acre, Cruzeiro do Sul, soil in a grassland in a tropical rain forest, 2001 Peru, Lima, human cornea, < 1995 Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis in a 3.5-year-old DSH cat, MN, 2008 Spain, human oropharyngeal exudate, 2004 Spain, human sputum, 2010 Portugal, bronchoalveolar lavage, chronic invasive aspergillosis in a 56-year-old male, 2007 Japan, human, sputum, 2011 Japan, abscess near thigh bone, 40-year-old man with osteomyelitis, 2012 Japan, human, clinical material, < 2007 Australia, thoracic mass in a cat, < 2005 Australia, retrobulbar mass, sino-orbital aspergillosis in a cat, < 2005 Czech Republic, near Kladno, soil of spoil-bank, 1993 Czech Republic, Markovičky, near Kutná Hora, old silver mine waste dump, 2007 Czech Republic, Chvaletice, soil crust, abandoned tailing pond, 2007 USA, Wyoming, Glenrock, soil from coal mine dump, 2010 Spain, Andalusia, Aracena, Gruta de la Maravillas, cave air, 2010 USA, Wyoming, Glenrock, soil from coal mine dump, 2010 Czech Republic, Krušné hory, near Abertamy, soil from old dump, 2011 USA, Wyoming, Glenrock, soil from coal mine dump, 2010 USA, Wyoming, Glenrock, soil from coal mine dump, 2010 Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 7-year-old DSH cat, FN, 2007 Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 3-year-old Himalayan cat, FN, 2009 Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis, 2-year-old Himalayan cat, MN, 2007 Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis, 5-year-old cat, Ragoll, MN, 2013 Australia, Canberra, retrobulbar mass, sino-orbital aspergillosis, 8-year-old domestic longhair cat Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 5-year-old DSH cat, FN, 2010 Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis, 2-year-old BSH cat, MN, 2012 Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 4-year-old DSH cat, FN, 2010 Australia, Sydney, vitreous humor, disseminated invasive apsergillosis 9-year-old Old English Sheepdog, MN, 2005 Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 4.5-year-old Ragdoll cat, MN, 2009 Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis, 2-year-old DSH cat, FN, 2007

MAT1-1-1 MAT1-1-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-1-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 MAT1-1-1 MAT1-1-1 MAT1-1-1

MAT1-1-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 MAT1-1-1

Brazil, Acre, Xapuri, grassland soil in cattle farm, 2001 Brazil, Amazonas, Manaus, tropical rain forest soil, 2001 Romania, Movile cave, above the Lake Room, cave sediment, 2014 Romania, Movile cave, cave sediment, 2014 Romania, Movile cave, Lake Room, cave sediment, 2014 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 Brazil, Pernambuco, near Arcoverde, semi-desert soil in a caatinga area, 2011 China, soil, 2008 China, soil, 2008 Australia, New South Wales, Warrumbungle National Park, sandy soil, 1971 Ghana, Tafo, soil, 1950

Aspergillus acrensis IFM 57291T = CCF 4670T (01-BA-462-5) IFM 57290 = CCF 4666 (01-BA-666-5) CCF 4959 (S973) CCF 4960 (S974) CCF 4961 (S975) A. arcoverdensis IFM 61334T = JCM 19878T = CCF 4900T (6-2-32) IFM 61333 = CCF 4899 (10-2-3) IFM 61337 = JCM 19879 = CCF 4901 (1-1-34) IFM 61338 = JCM 19880 = CCF 4902 (6-2-3) IFM 61339 = CCF 4903 (2-1-11) IFM 61340 = CCF 4904 (7-2-33) IFM 61345 = CCF 5633 (3-2-2) IFM 61346 = CCF 4906 (4-2-14) IFM 61349 = CCF 4907 (4-2-9) IFM 61362 = CCF 4908 (5-2-2) IFM 59922 = CCF 4560 (08-SA-2-2) IFM 59923 = CCF 4569 (08-SA-2-1) FRR 1266 = CBS 121595 = DTO 019-F2 = CCF 4574 A. aureolus IFM 47021T = IFM 46935T = IFM 53589T = CBS 105.55T = NRRL 2244T = IMI 06145T = KACC 41204T = KACC 41095T = CCF 4644T = CCF 4646T = CCF 4648T IFM 46584 = IFM 46936 = CBM-FA-0692 = CCF 4645 = CCF 4647 IFM 53615 = CBM-FA-934 = CCF 4571 (ex-type of A. indohii) IHEM 22515 (RV 71215) A. felis CBS 130245T = DTO 131-F4T = CCF 5620 NRRL 62900 = CM-3147 = CCF 4895 (ex-type of A. parafelis) NRRL 62903 = CM-6087 = CCF 4897 (ex-type of A. pseudofelis) NRRL 62901 = CM-5623 = CCF 4896 = CCF 4557 (Viridi-Pinh) IFM 59564 = CCF 5612 IFM 60053 = CCF 4559 IFM 54303 = CCF 4570 FRR 5679 = CCF 5613 (MK246) FRR 5680 = CCF 5615 (MK284) CCF 2937 CCF 4002 (AK 196/07) CCF 4003 (AK 27/07) CCF 4171 = CMF ISB 2162 = IFM 60852 (F39) CCF 4172 (F47) CCF 4148 = CMF ISB 1975 = IFM 60868 (F22) CCF 4376 (AK 102/11) CCF 4497 = CMF ISB 1936 (F6) CCF 4498 = IFM 60853 (F49) DTO 131-E4 = CCF 5609 (2384/07) DTO 131-E5 = CCF 5610 (4091/09) DTO 131-G1 = CCF 5611 (834/07) CCF 5614 (14/4138) CCF 5616 (Felix H. D) DTO 131-F1 = CCF 5617 (66/10) CCF 5618 (Luigi C.) CBS 130248 = DTO 131-G3 = CCF 5619 (1767/10) CBS 130249 = DTO 155-G3 = CCF 5621 (1207/05) DTO 131-F2 = CCF 5622 (3532/09) CBS 130247 = DTO 131-G2 = CCF 5623 (1020/07)

MAT locus 4,5

Locality, substrate, year of isolation3

Species / Culture collection nos. 1,2

Table 1   List of Aspergillus strains, information on isolation source and reproductive strategy.

V. Hubka et al.: Species delimitation and hybridization in section Fumigati

143

Locality, substrate, year of isolation3

A. felis (cont.) DTO 131-E9 = CCF 5624 (1848/08) Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 1.5-year-old DSH cat, MN, 2008 DTO 131-E3 = CCF 5625 (3008/08 D) Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 8-year-old Persian cat, FN, 2008 DTO 131-F6 = CCF 5626 (8651/09) Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 8-year-old DSH cat, MN, 2009 CBS 130244 = DTO 131-E6 = CCF 5627 (4067/09D) Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis, 5-year-old Cornish Rex cat, FN, 2009 DTO 131-F3 = CCF 5628 (2188/08) Australia, Brisbane, retrobulbar mass, sino-orbital aspergillosis, 7-year-old DSH cat, FN, 2008 CBS 130246 = DTO 131-F9 = CCF 5629 (448/08) Australia, Sydney, nasal cavity, sino-nasal aspergillosis 13-year-old DLH cat, MN, 2008 A. frankstonensis CBS 142233T = IBT 34172T = DTO 341-E7T = CCF 5799T Australia, Victoria, Frankston, woodland soil, 2015 CBS 142234 = IBT 34204 = DTO 341-F3 = CCF 5798 Australia, Victoria, Frankston, woodland soil, 2015 A. pseudoviridinutans T NRRL 62904 = CCF 5631 (NIHAV1, 1720) USA, U.S. National Institutes of Health, mediastinal lymph node, 14-year-old boy with chronic granulomatous disease, 2004 CBS 458.75 = KACC 41203 = IHEM 9862 (ex-type of India, Lucknow, Mohanlalganj, soil, < 1971 A. fumigatus var. sclerotiorum) IMI 182127 = KACC 41614 = CCF 5630 Srí Lanka, Pinus caribea, < 1974 IFM 55266 = CCF 5644 Japan, human, lung, 2004 IFM 57289 = CCF 4665 Brazil, Mato Grosso, soil IFM 59502 = CCF 4561 Japan, cornea, keratomycosis, 26-year-old woman, 2011 IFM 59503 = CCF 4562 Japan, cornea, keratomycosis, 26-year-old woman, 2011 CCF 5632 (NIHAV2, 2594) USA, lung biopsy, 8-year-old boy with hyperimmunoglobulin-E syndrome, 2004 A. siamensis T T T IFM 59793 = KUFC 6349 = CCF 4685 Thailand, Chonburi Province, Samaesarn Island, coastal forest soil, 2008 IFM 61157 = KUFC 6397 = CCF 4686 Thailand, Chiang Mai, termite nest soil, 2009 A. udagawae   IFM 46972T = CBS 114217T = DTO 157-D7T = CBM-FA 0702T = Brazil, São Paulo State, Botucatú, Lagoa Seka Avea, plantation soil, 1993 KACC 41155T = CCF 4558T IFM 46973 = CBS 114218 = DTO 157-D8 = CBM-FA 0703 = Brazil, São Paulo State, Botucatú, Lagoa Seka Avea, plantation soil, 1993 KACC 41156 = CCF 5672 IFM 5058 = CCF 4662 Japan, human, eye IFM 51744 = CCF 4671 Japan, human, clinical material, 2002 IFM 53868 = CCF 4667 Japan, human, clinical material, 2004 IFM 54131 = CBM-FA-0697 = CCF 4663 China, Shaanxi, soil, 1994 IFM 54132 = CBM-FA-0698 = CCF 4664 China, Shaanxi, soil, 1994 IFM 54745 = CBM-FA-694 = CCF 4661 China, Shaanxi, soil, 1994 IFM 55207 = NBRC 31952 = CCF 4660 Russia, soil, 1985 IFM 62155 = CCF 4668 Brazil, soil, 2008 CCF 4475 (F2) USA, Wyoming, Glenrock, prairie soil, 2010 CCF 4476 (F32) USA, Wyoming, Glenrock, soil, mine waste dump, 2010 CCF 4478 = CMF ISB 2193 (F66) USA, Wyoming, Gilette, soil, mine waste dump, 2011 CCF 4479 = CMF ISB 2189 (F70) USA, Illinois, soil, mine waste dump, 2011 CCF 4481 = CMF ISB 2191 (F83) USA, Wyoming, Gilette, soil, mine waste dump, 2011 CCF 4491 = CMF ISB 1971 (F3) USA, Wyoming, Glenrock, prairie soil, 2010 CCF 4492 (F21) USA, Wyoming, Glenrock, soil, mine waste dump, 2010 CCF 4494 (F44) USA, Wyoming, Glenrock, prairie soil, 2010 CMF ISB 1972 = CCF 4502 (F11) USA, Wyoming, Glenrock, soil, mine waste dump, 2010 CMF ISB 2190 = CCF 5635 (F76) USA, Indiana, soil, mine waste dump, 2011 CMF ISB 2509 = CCF 5636 (F20) USA, Wyoming, Glenrock, soil, mine waste dump, 2010 CCF 5637 (F37) USA, Wyoming, Gilette, soil, mine waste dump, 2008 CCF 5638 (3C8) USA, Philadelphia, retrobulbar mass, sino-orbital aspergillosis, 4-year-old Persian cat, MN, 2012 DTO 166-D6 = CCF 5639 (11.3356, Milo) Australia, Sydney, retrobulbar mass, sino-orbital aspergillosis 2-year-old DSH cat, MN, 2011 CCF 5634 (B3) Czech Republic, Hostěradice, earthworm casts, 2012 A. viridinutans   T T T T T IFM 47045 = IFM 47046 = IMI 367415 = IMI 062875 = NRRL 4365 = Australia, Victoria, Frankston, rabbit dung, 1954 T T T T T NRRL 576 = CBS 127.56 = KACC 41142 = CCF 4382 = CCF 4568 A. wyomingensis   CCF 4417T = CMF ISB 2494T = CBS 135456T (F30) USA, Wyoming, Glenrock, soil, mine waste dump, 2010 CCF 4169 = CMF ISB 2486 (F24) USA, Wyoming, Glenrock, soil, 2010

Species / Culture collection nos. 1,2

Table 1   (cont.)

MAT1-1-1 (HF937391) MAT1-1-1

MAT1-1-1 (HF937390)

MAT1-1-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 MAT1-2-1 (HF937389) MAT1-2-1 MAT1-2-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-1-1 ND MAT1-2-1

MAT1-2-1

MAT1-1-1

homothallic homothallic

MAT1-2-1 MAT1-1-1 MAT1-2-1 MAT1-1-1 MAT1-1-1 MAT1-1-1 (LT796761)

MAT1-1-1 (KJ858509) MAT1-2-1

MAT1-2-1 MAT1-2-1

MAT1-1-1 (KC797628) MAT1-1-1 (KC797634) MAT1-2-1 (KC797624) MAT1-1-1 (KC797630) MAT1-2-1 MAT1-1-1 (KC797631)

MAT locus 4,5

144 Persoonia – Volume 41, 2018

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MAT1-2-1

Culture collection acronyms: CBM-FA = Natural History Museum & Institute, Chiba, Japan; CBS = CBS culture collection housed at the Westerdijk Institute, Utrecht, The Netherlands; CCF = Culture Collection of Fungi, Prague, Czech Republic; CM = Filamentous fungus collection of the Spanish National Center for Microbiology, Madrid, Spain; CMF ISB = Collection of Microscopic Fungi, Institute of Soil Biology, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic; DTO = working collection of the Applied and Industrial Mycology department housed at the Westerdijk Institute, Utrecht, The Netherlands; FRR = Food Fungal Culture Collection, North Ryde, Australia; IBT = culture collection of the DTU Systems Biology, Lyngby, Denmark; IFM = Collection at the Medical Mycology Research Centre, Chiba University, Japan; IHEM = Belgian Coordinated Collections of Micro-organisms (BCCM/IHEM), Brussels, Belgium; IMI = CABI’s collection of fungi and bacteria, Egham, UK; JCM = Japan Collection of Microorganisms, Tsukuba, Japan; KACC = Korean Agricultural Culture Collection, Wanju, South Korea; KUFC = Kasetsart University Fungal Collection, Bangkok, Thailand; NBRC (IFO) = Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan; NRRL = Agricultural Research Service Culture Collection, Peoria, Illinois, USA. 2 Original numbers of strains and personal strain designations are given in parentheses. 3 BSH = British shorthair; DLH = domestic longhair; DSH = domestic shorthair; FN = female neutered (desexed); MN = male neutered; ND = not determined. 4 When available, sequence number in public database is given in parentheses; in the remaining cases, the MAT idiomorph was confirmed only on the electrophoretogram (specific PCR and length of amplicons). 5 Sequences generated in this study are in bold. 1

USA, Wyoming, Glenrock, soil, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 USA, Wyoming, Glenrock, soil, mine waste dump, 2010 Russia, Moscow, soil, < 1968 China, Urumqi, soil, 2008 Australia, Melbourne, retrobulbar mass in a 1.5-year-old BSH cat, MN, 2010 USA, human, clinical material A. wyomingensis (cont.) CCF 4170 = CMF ISB 2485 (F12) CCF 4411 = CMF ISB 1977 = IFM 60854 (F5) CCF 4412 (F9) CCF 4413 = CMF ISB 2317 (F10) CCF 4414 = CMF ISB 1974 = IFM 60856 (F13) CCF 4415 = CMF ISB 2487 (F28) CCF 4416 = CMF ISB 1976 = CBS 135455 (F29) CCF 4418 = CMF ISB 2162 = IFM 60855 (F31) CCF 4419 = CMF ISB 2495 (F53) CCF 4420 = CMF ISB 2491 (F60) IMI 133982 = CCF 4383 IFM 59681 = CCF 4563 DTO 155-G2 = CCF 5640 (Yogurt R.) outgroup A. lentulus NRRL 35552T = CBS 117885T = IBT 27201T = KACC 41940T

MAT1-2-1 (LT796765) MAT1-2-1 MAT1-1-1 MAT1-1-1 MAT1-1-1 (LT796762) MAT1-1-1 MAT1-2-1 (HF937388) MAT1-2-1 MAT1-2-1 MAT1-1-1 MAT1-1-1 (LT796763) MAT1-2-1 (LT796764) MAT1-2-1

Locality, substrate, year of isolation3 Species / Culture collection nos. 1,2

Table 1   (cont.)

MAT locus 4,5

V. Hubka et al.: Species delimitation and hybridization in section Fumigati

Homothallism is a predominant reproductive mode in sect. Fumigati and many species readily produce ascomata (neosartorya-morph) in culture, while others are heterothallic or have an unknown sexual morph (Hubka et al. 2017). Homothallic species are infrequently pathogenic, although A. thermomutatus is a notable exception. The majority of clinically relevant species belong to the A. fumigatus clade (Balajee et al. 2005b, 2009, Yaguchi et al. 2007, Alcazar-Fuoli et al. 2008) or the AVSC (Sugui et al. 2010, 2014, Barrs et al. 2013, Nováková et al. 2014) and are heterothallic. A cryptic sexual cycle of several of these opportunistic pathogens, including A. fumigatus (O’Gorman et al. 2009), A. lentulus (Swilaiman et al. 2013) and A. felis (Barrs et al. 2013), was discovered recently by crossing opposite mating type isolates in vitro. Molecular methods are routinely used for identification of spe­ cies from sect. Fumigati due to overlapping morphological features of their asexual morph. In contrast, the morphology of the sexual morph, especially of ascospores, is amongst the most informative of phenotypic characteristics in sect. Fumigati. The taxonomy of AVSC has developed rapidly since eight of the currently 11 recognized species were described in the last four years (Barrs et al. 2013, Eamvijarn et al. 2013, Nováková et al. 2014, Sugui et al. 2014, Matsuzawa et al. 2015, Talbot et al. 2017). The species boundaries delimitation was usually based on comparison of single-gene phylogenies and principles of genealogical concordance. In addition, some studies supported the species concept by results of in vitro mating experiments between opposite mating type strains. With the increasing number of species, available isolates and new mating experiment data, the species boundaries in AVSC became unclear as pointed out by Talbot et al. (2017) who used the designation ‘A. felis clade’ for A. felis and related species. Importantly, Sugui et al. (2014) and Talbot et al. (2017) identified that interpretation of in vitro mating assays in sect. Fumigati may be problematic because different phylogenetic species in the AVSC were able to produce fertile ascomata when crossed between themselves. Some even mated successfully with A. fumigatus s.str. Here we present a critical re-evaluation of species boundaries in the AVSC. We examined a large set of clinical and environmental strains collected worldwide. We did not use classical phylo­ genetic methods or genealogical concordance phylogenetic species recognition rules (GCPSR) for species delimitation due to their unsatisfactory results in previous AVSC studies. Such methods, based predominantly on analysis of concatenated DNA sequence data or comparison of single-gene phylogenies are frequently prone to species over-delimitation or are affected by subjective judgements of species boundaries. Instead, we used recently introduced delimitation techniques based on coalescent theory and the multispecies coalescent model (MSC) (Flot 2015). We followed the approach recommended by Carstens et al. (2013) that combines species delimitation, species tree estimation and species validation steps. Although these methods have already been applied to other groups of organisms such as animals and plants their use in fungi is scarce (Stewart et al. 2014, Singh et al. 2015, Liu et al. 2016, Sklenář et al. 2017, Hubka et al. 2018). Here, the results of MSC methods were taken as a basic hypothesis for species delimitation and then further verified by analysis of intra- and interspecific biological compatibilities, as well as ascospore dimensions and ornamentation. MATERIAL AND METHODS Fungal strains A total of 110 isolates were examined including new isolates and isolates obtained from previously published studies (Katz et



Culture collection nos.1 ITS

GenBank /ENA /DDBJ accession numbers benA CaM RPB2 act

mcm7 tsr1

Persoonia – Volume 41, 2018

Aspergillus acrensis

T

T

IFM 57291 = CCF 4670 – LT795980 LT795981 LT795982 LT795983 – – IFM 57290 = CCF 4666 – LT795976 LT795977 LT795978 LT795979 – – CCF 4959 – LT795984 LT558741 LT795985 LT795986 – – CCF 4960 – LT795987 LT558742 LT795988 LT795989 – – CCF 4961 – LT795990 LT558743 LT795991 LT795992 – – A. arcoverdensis IFM 61334T = JCM 19878T = CCF 4900T – AB818845 LT795958 LT795959 AB818867 – – IFM 61333 = CCF 4899 – LT795954 LT795955 LT795956 LT795957 – – IFM 61337 = JCM 19879 = CCF 4901 – AB818846 LT795960 LT795961 AB818868 – – IFM 61338 = JCM 19880 = CCF 4902 – AB818847 LT795962 LT795963 AB818869 – – IFM 61339 = CCF 4903 – AB818848 LT795964 LT795965 AB818870 – – IFM 61340 = CCF 4904 – AB818849 LT795966 LT795967 AB818871 – – IFM 61345 = CCF 5633 – AB818850 LT795968 LT795969 AB818872 – – IFM 61346 = CCF 4906 – AB818851 LT795970 LT795971 AB818873 – – IFM 61349 = CCF 4907 – AB818852 LT795972 LT795973 AB818874 – – IFM 61362 = CCF 4908 – AB818853 LT795974 LT795975 AB818875 – – IFM 59922 = CCF 4560 – LT795944 LT795945 LT795946 LT795947 – – IFM 59923 = CCF 4569 – AB818844 LT795948 LT795949 AB818866 – – FRR 1266 = CBS 121595 = DTO 019-F2 = CCF 4574 JX021672 LT795950 LT795951 LT795952 LT795953 – – A. aureolus IFM 47021T = IFM 46935T = IFM 53589T = CBS 105.55T = NRRL 2244T = IMI 06145T = KACC 41204T = EF669950 EF669808 HG426051 EF669738 DQ094861 KJ914718 KJ914750 KACC 41095T = CCF 4644T = CCF 4646T = CCF 4648T IFM 46584 = IFM 46936 = CBM-FA-0692 = CCF 4645 = CCF 4647 – LT796001 HG426050 LT796002 LT796003 – – IFM 53615 = CBM-FA-934 = CCF 4571 (ex-type of A. indohii ) – AB488757 LT795998 LT795999 LT796000 – – IHEM 22515 – LT796004 LT796005 LT796006 LT796007 LT796153 LT796756 A. felis CBS 130245T = DTO 131-F4T = CCF 5620 KF558318 KJ914694 KJ914706 KJ914735 LT795880 KJ914724 LT796745 NRRL 62900 = CM-3147 = CCF 4895 (ex-type of A. parafelis) – KJ914692 KJ914702 LT795839 LT795838 KJ914720 LT796734 NRRL 62903 = CM-6087 = CCF 4897 (ex-type of A. pseudofelis) – KJ914697 KJ914705 LT795891 LT795892 KJ914723 LT796749 NRRL 62901 = CM-5623 = CCF 4896 = CCF 4557 – KJ914693 LT795813 LT795814 LT795815 LT796152 LT796727 IFM 59564 = CCF 5612 – LT795801 LT795802 LT795803 LT795804 LT796126 LT796724 IFM 60053 = CCF 4559 – LT795856 LT795857 LT795858 LT795859 LT796138 LT796739 IFM 54303 = CCF 4570 AB250780 LT795860 LT795861 LT795862 LT795863 LT796139 LT796740 FRR 5679 = CCF 5613 – LT795805 LT795806 LT795807 LT795808 LT796127 LT796725 FRR 5680 = CCF 5615 – LT795844 LT795845 LT795846 LT795847 LT796135 LT796736 CCF 2937 – LT795816 LT795817 LT795818 LT795819 LT796129 LT796728 CCF 4002 FR733865 FR775350 LT795824 LT795825 LT795826 LT796131 LT796730 CCF 4003 FR733866 FR775349 LT795827 LT795828 LT795829 LT796132 LT796731 CCF 4171 = CMF ISB 2162 = IFM 60852 – LT795840 LT795841 LT795842 LT795843 LT796134 LT796735 CCF 4172 – LT795834 LT795835 LT795836 LT795837 LT796133 LT796733 CCF 4148 = CMF ISB 1975 = IFM 60868 HE578063 LT795868 LT795869 LT795870 LT795871 – LT796741 CCF 4376 – LT795872 LT795873 LT795874 LT795875 LT796141 LT796743 CCF 4497 = CMF ISB 1936 – LT795820 LT795821 LT795822 LT795823 LT796130 LT796729 CCF 4498 = IFM 60853 – LT795830 LT795831 LT795832 LT795833 – LT796732 DTO 131-E4 = CCF 5609 JX021673 LT795789 LT795790 LT795791 LT795792 LT796123 LT796721 DTO 131-E5 = CCF 5610 JX021674 LT795793 LT795794 LT795795 LT795796 LT796124 LT796722 DTO 131-G1 = CCF 5611 JX021682 LT795797 LT795798 LT795799 LT795800 LT796125 LT796723 CCF 5614 – LT795809 LT795810 LT795811 LT795812 LT796128 LT796726 CCF 5616 – LT795848 LT795849 LT795850 LT795851 LT796136 LT796737 DTO 131-F1 = CCF 5617 JX021677 LT795852 LT795853 LT795854 LT795855 LT796137 LT796738 CCF 5618 – LT795864 LT795865 LT795866 LT795867 LT796140 LT796742 CBS 130248 = DTO 131-G3 = CCF 5619 JX021684 LT795876 LT795877 LT795878 LT795879 LT796142 LT796744

Species

Table 2   List of Aspergillus strains and sequences used in phylogenetic analysis; accession numbers in bold were generated for this study.

146



Culture collection nos.1

CBS 130249 = DTO 155-G3 = CCF 5621 DTO 131-F2 = CCF 5622 CBS 130247 = DTO 131-G2 = CCF 5623 DTO 131-E9 = CCF 5624 DTO 131-E3 = CCF 5625 DTO 131-F6 = CCF 5626 CBS 130244 = DTO 131-E6 = CCF 5627 DTO 131-F3 = CCF 5628 CBS 130246 = DTO 131-F9 = CCF 5629 A. frankstonensis CBS 142233T = IBT 34172T = DTO 341-E7T = CCF 5799T CBS 142234 = IBT 34204 = DTO 341-F3 = CCF 5798 A. pseudoviridinutans NRRL 62904T = CCF 5631 CBS 458.75 = KACC 41203 = IHEM 9862 (ex-type of A. fumigatus var. sclerotiorum) IMI 182127 = KACC 41614 = CCF 5630 IFM 55266 = CCF 5644 IFM 57289 = CCF 4665 IFM 59502 = CCF 4561 IFM 59503 = CCF 4562 CCF 5632 A. siamensis IFM 59793T = KUFC 6349T = CCF 4685T IFM 61157 = KUFC 6397 = CCF 4686 A. udagawae IFM 46972T = CBS 114217T = DTO 157-D7T = CBM-FA 0702T = KACC 41155T = CCF 4558T IFM 46973 = CBS 114218 = DTO 157-D8 = CBM-FA 0703 = KACC 41156 = CCF 5672 IFM 5058 = CCF 4662 IFM 51744 = CCF 4671 IFM 53868 = CCF 4667 IFM 54131 = CBM-FA-0697 = CCF 4663 IFM 54132 = CBM-FA-0698 = CCF 4664 IFM 54745 = CBM-FA-694 = CCF 4661 IFM 55207 = NBRC 31952 = CCF 4660 IFM 62155 = CCF 4668 CCF 4475 CCF 4476 CCF 4478 = CMF ISB 2193 CCF 4479 = CMF ISB 2189 CCF 4481 = CMF ISB 2191 CCF 4491 = CMF ISB 1971 CCF 4492 CCF 4494 CMF ISB 1972 = CCF 4502 CMF ISB 2190 = CCF 5635 CMF ISB 2509 = CCF 5636 CCF 5637 CCF 5638 DTO 166-D6 = CCF 5639 CCF 5634 A. viridinutans IFM 47045T = IFM 47046T = IMI 367415T = IMI 062875T = NRRL 4365T = NRRL 576T = CBS 127.56T = KACC 41142T = CCF 4382T = CCF 4568T

A. felis (cont.)

Species

Table 2   (cont.)

JX021686 JX021678 JX021683 JX021676 JX021671 JX021680 JX021675 JX021679 JX021681 KY808756 KY808761 – – – – – – – – – – AB185265 JN943591 AB250402 AB250403 AB250405 – – – – – – – – – – – – – HE578061 – – – – – – EF669978

ITS

GenBank /ENA /DDBJ accession numbers mcm7 tsr1

JX021711 JX021713 LT795881 LT795882 LT796143 LT796746 LT795883 LT795884 LT795885 LT795886 LT796144 LT796747 LT795887 LT795888 LT795889 LT795890 LT796145 LT796748 LT795893 LT795894 LT795895 LT795896 LT796146 LT796750 LT795897 LT795898 LT795899 LT795900 LT796147 LT796751 LT795901 LT795902 LT795903 LT795904 LT796148 LT796752 LT795905 LT795906 LT795907 LT795908 LT796149 LT796753 LT795909 LT795910 LT795911 LT795912 LT796150 LT796754 LT795913 LT795914 LT795915 LT795916 LT796151 LT796755 KY808594 KY808724 KY808948 KY808549 KY808901 LT904842 KY808599 KY808729 KY808953 KY808554 KY808906 – KJ914690 KJ914708 LT795930 LT795931 LT796119 LT796717 LT795925 HG426048 LT795926 DQ094853 LT796117 LT796715 LT795927 LT795928 LT795929 DQ094850 LT796118 LT796716 LT795917 LT795918 LT795919 LT795920 LT796115 LT796713 LT795921 LT795922 LT795923 LT795924 LT796116 LT796714 LT795936 LT795937 LT795938 LT795939 LT796121 LT796719 LT795940 LT795941 LT795942 LT795943 LT796122 LT796720 LT795932 LT795933 LT795934 LT795935 LT796120 LT796718 AB646989 LT795993 LT795994 AB776703 – – AB776701 LT795995 LT795996 LT795997 – – LT796063 LT796064 LT796065 LT796066 – – LT796067 LT796068 LT796069 LT796070 – – LT796075 LT796076 LT796077 LT796078 – – LT796079 LT796080 LT796081 LT796082 – – LT796111 LT796112 LT796113 LT796114 – – LT796083 LT796084 LT796085 LT796086 – – LT796087 LT796088 LT796089 LT796090 – – LT796091 LT796092 LT796093 LT796094 – – LT796095 LT796096 LT796097 LT796098 – – LT796099 LT796100 LT796101 LT796102 – – HF933366 HF933407 LT796037 LT796038 – – HF933371 HF933412 LT796043 LT796044 – – HF933376 HF933416 LT796045 LT796046 – – HF933377 HF933417 LT796047 LT796048 – – HF933379 HF933419 LT796049 LT796050 – – HF933370 HF933411 LT796051 LT796052 – – HF933368 HF933409 LT796053 LT796054 – – HF933373 HF933413 LT796055 LT796056 – – HE578075 HF933405 LT796057 LT796058 – – HG426055 HG426049 LT796059 LT796060 – – HF933367 HF933408 LT796061 LT796062 – – LT796071 LT796072 LT796073 LT796074 – – LT796103 LT796104 LT796105 LT796106 LT796156 LT796758 LT796107 LT796108 LT796109 LT796110 LT796155 LT796759 LT796039 LT796040 LT796041 LT796042 – – EF669834 EF669904 EF669765 DQ094862 KJ914717 KJ914751

benA CaM RPB2 act

V. Hubka et al.: Species delimitation and hybridization in section Fumigati

147

148

KJ914746 EF669895

EF669756

DQ094873

KJ914712 EF669825 EF669969 NRRL 35552T = CBS 117885T = IBT 27201T = KACC 41940T

outgroup A. lentulus

1

HF933359 HF933397 HF937378 HF937382 – – HF933354 HF933394 LT796009 LT796008 – – HF933356 HF933392 LT796011 LT796010 – – HE578077 HF933389 LT796016 LT796015 – – HF933352 HF933390 LT796018 LT796017 – – HF933360 HF933391 LT796019 LT796020 – – HF933353 HF933393 LT796021 LT796022 – – HF933357 HF933395 LT796023 LT796024 – – HF933358 HF933396 HF937377 HF937381 – – HF933355 HF933398 LT796025 LT796026 – – HF933361 HF933399 LT796027 LT796028 – – HF933362 HF933400 LT796029 LT796030 – – LT796012 LT796013 LT796014 DQ094860 – – HG426056 HG426053 LT796031 LT796032 – – LT796033 LT796034 LT796035 LT796036 LT796154 LT796757 HG324081 – – HE578062 – – – – – – – – – – – CCF 4417T = CMF ISB 2494T = CBS 135456T CCF 4169 = CMF ISB 2486 CCF 4170 = CMF ISB 2485 CCF 4411 = CMF ISB 1977 = IFM 60854 CCF 4412 CCF 4413 = CMF ISB 2317 CCF 4414 = CMF ISB 1974 = IFM 60856 CCF 4415 = CMF ISB 2487 CCF 4416 = CMF ISB 1976 = CBS 135455 CCF 4418 = CMF ISB 2162 = IFM 60855 CCF 4419 = CMF ISB 2495 CCF 4420 = CMF ISB 2491 IMI 133982 = CCF 4383 IFM 59681 = CCF 4563 DTO 155-G2 = CCF 5640 A. wyomingensis

GenBank /ENA /DDBJ accession numbers

mcm7 tsr1 benA CaM RPB2 act ITS

Culture collection nos.1 Species

Table 2   (cont.)

Culture collection acronyms: CBM-FA = Natural History Museum & Institute, Chiba, Japan; CBS = CBS culture collection housed at the Westerdijk Institute, Utrecht, The Netherlands; CCF = Culture Collection of Fungi, Prague, Czech Republic; CM = Filamentous fungus collection of the Spanish National Center for Microbiology, Madrid, Spain; CMF ISB = Collection of Microscopic Fungi, Institute of Soil Biology, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic; DTO = working collection of the Applied and Industrial Mycology department housed at the Westerdijk Institute, Utrecht, The Netherlands; FRR = Food Fungal Culture Collection, North Ride, Australia; IBT = culture collection of the DTU Systems Biology, Lyngby, Denmark; IFM = Collection at the Medical Mycology Research Centre, Chiba University, Japan; IHEM = Belgian Coordinated Collections of Micro-organisms (BCCM/IHEM), Brussels, Belgium; IMI = CABI’s collection of fungi and bacteria, Egham, UK; JCM = Japan Collection of Microorganisms, Tsukuba, Japan; KACC = Korean Agricultural Culture Collection, Wanju, South Korea; KUFC = Kasetsart University Fungal Collection, Bangkok, Thailand; NBRC (IFO) = Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan; NRRL = Agricultural Research Service Culture Collection, Peoria, Illinois, USA.

Persoonia – Volume 41, 2018

al. 2005, Vinh et al. 2009, Coelho et al. 2011, Shigeyasu et al. 2012, Barrs et al. 2013, 2014, Eamvijarn et al. 2013, Nováková et al. 2014, Sugui et al. 2014, Matsuzawa et al. 2015, Talbot et al. 2017) and culture collections. The set comprised 38 clinical strains and 72 environmental isolates, including 67 from soil, four from cave environments and one from plant material. The provenance of isolates is detailed in Table 1. Newly isolated strains were deposited into the Culture Collection of Fungi at the Department of Botany, Charles University, Prague, Czech Republic (CCF). Dried herbarium specimens were deposited into the herbaria of the Medical Mycology Research Center, Chiba University, Japan (IFM) and Mycological Department of the National Museum, Prague, Czech Republic (PRM). Phenotypic studies The strains were grown on malt extract agar (MEA), Czapek Yeast Autolysate Agar (CYA), Czapek-Dox agar (CZA), yeast extract sucrose agar (YES), CYA supplemented with 20 % sucrose (CY20S), and creatine sucrose agar (CREA), and incubated at 25 °C. Agar media composition was based on that described by Samson et al. (2014). Malt extract and yeast extract were obtained from Oxoid (Basingstoke, UK) and Fluka Chemie GmbH (Switzerland), respectively. Growth at 42, 45 and 47 °C was tested on MEA plates sealed with Parafilm. Colour determination was performed according to the ISCC-NBS (InterSociety Color Council – National Bureau of Standards) Centroid Colour Charts (Kelly 1964). Micromorphology was observed on MEA. Lactic acid with cotton blue was used as a mounting medium. Photographs were taken on an Olympus BX-51 microscope (Olympus DP72 camera) using Nomarski contrast. Macromorphology of the colonies was documented using a stereomicroscope Olympus SZ61 (with Olympus Camedia C-5050 Zoom camera) or Canon EOS 500D. Scanning electron microscopy (SEM) was performed using a JEOL-6380 LV scanning electron microscope (JEOL Ltd. Tokyo, Japan) as described by Hubka et al. (2013b). Briefly, pieces of colony or mature ascomata were fixed in osmium tetroxide vapours for one wk at 5–10 °C and gold coated using a Bal-Tec SCD 050 sputter coater. The specimens were observed using 40 μm spot size and 15 – 25 kV accelerating voltage. Molecular studies ArchivePure DNA yeast and Gram2+ kit (5 PRIME Inc., Gaithersburg, MD) was used for DNA isolation from 7-d-old cultures according to the manufacturer’s instructions as updated by Hubka et al. (2015b). The purity and concentration of extracted DNA was evaluated by NanoDrop 1000 Spectrophotometer. ITS rDNA region was amplified using forward primers ITS1 or ITS5 (White et al. 1990) and reverse primers ITS4S (Kretzer et al. 1996) or NL4 (O’Donnell 1993); partial β-tubulin gene (benA) using forward primers Bt2a (Glass & Donaldson 1995) or Ben2f (Hubka & Kolařík 2012) and reverse primer Bt2b (Glass & Donaldson 1995); partial calmodulin gene (CaM) using forward primers CF1M or CF1L and reverse primer CF4 (Peterson 2008); partial actin gene (act) using primers ACT-512F and ACT-783R (Carbone & Kohn 1999); partial RNA polymerase II second largest subunit (RPB2) using forward primers fRPB2-5F (Liu et al. 1999) or RPB2-F50-CanAre (Jurjević et al. 2015) and reverse primer fRPB2-7cR (Liu et al. 1999); partial mcm7 gene encoding minichromosome maintenance factor 7 with primers Mcm7-709for and Mcm7-1348rev (Schmitt et al. 2009); and partial tsr1 gene encoding ribosome biogenesis protein with primers Tsr1-1453for and Tsr1-2308rev (Schmitt et al. 2009). Terminal primers were used for sequencing. The PCR reaction volume of 20 µL contained 1 µL (50 ng) of DNA, 0.3 µL of both primers (25 pM/mL), 0.2 µL of MyTaq TM DNA

149

V. Hubka et al.: Species delimitation and hybridization in section Fumigati

Polymerase (Bioline, GmbH, Germany) and 4 μL of 5 × MyTaq PCR buffer. The ITS rDNA, benA and CaM fragments were amplified using the following thermal cycle profile: 93 °C/2 min; 30 cycles of 93 °C/30 s; 55 °C/30 s; 72 °C/60 s; 72 °C/10 min. The annealing temperature for amplification of act gene was 60 °C (30 cycles); and that for tsr1 gene 50 °C (37 cycles). Partial RPB2 gene fragments were amplified using the abovementioned cycle or touchdown thermal-cycling: 93 °C/2 min; 5 cycles of 93 °C/30 s, 65–60 °C/30 s, 72 °C/60 s; 38 cycles of 93 °C/30 s, 55 °C/30 s, 72 °C/60 s; 72 °C/10 min. The partial mcm7 gene was amplified using modified touchdown thermalcycling: 93 °C/2 min; 5 cycles of 93 °C/30 s, 65 – 60 °C/30 s, 72 °C/60 s; 38 cycles of 93 °C/30 s, 60 °C/30 s, 72 °C/60 s; 72 °C/10 min. PCR product purification followed the protocol of Réblová et al. (2016). Automated sequencing was performed at Macrogen Sequencing Service (Amsterdam, The Netherlands) using both terminal primers. Sequences were deposited into the ENA (European Nucleotide Archive) database under the accession numbers listed in Table 2. Phylogenetic analysis Sequences were inspected and assembled using Bioedit v. 7.2.5 (www.mbio.ncsu.edu/BioEdit /bioedit.html). Alignments of the benA, CaM, act and RPB2 regions were performed using the G-INS-i option implemented in MAFFT v. 7 (Katoh & Standley 2013). Alignments were trimmed, concatenated and then analysed using Maximum likelihood (ML) and Bayesian inference (BI) analyses. Suitable partitioning scheme and substitution models (Bayesian information criterion) for analyses were selected using the greedy algorithm implemented in PartitionFinder v. 1.1.1 (Lanfear et al. 2017) with settings allowing introns, exons and codon positions to be independent partitions. Proposed partitioning schemes and substitution models for each dataset are listed in Table 3. The alignment characteristics are listed in Table 4.

The ML tree was constructed with IQ-TREE v. 1.4.4 (Nguyen et al. 2015) with nodal support determined by non-parametric bootstrapping (BS) with 1 000 replicates. Bayesian posterior probabilities (PP) were calculated using MrBayes v. 3.2.6 (Ronquist et al. 2012). The analyses ran for 10 7 generations, two parallel runs with four chains each were used, every 1 000th tree was retained, and the first 25 % of trees were discarded as burn-in. The trees were rooted with Aspergillus clavatus NRRL 1 and A. lentulus NRRL 35552, respectively. All alignments are available from the Dryad Digital Repository (https://doi. org/10.5061/dryad.38889). Species delimitation and species tree inference Several species delimitation methods were applied to elucidate the species boundaries within the AVSC. We followed the recommendation of Carstens et al. (2013) and compared the results of several different methods. The analysis was divided into two parts. Four genetic loci were examined in the first analysis which comprised all species from the AVSC while six genetic loci were examined in the second analysis focused on the clade comprising Aspergillus felis, A. pseudofelis, A. para­ felis and A. pseudoviridinutans (A. aureolus was used as an outgroup). The alignment characteristics are listed in Table 4. Only unique nucleotide sequences, selected with DAMBE v. 6.4.11 (Xia 2017) were used in the analyses. Nucleotide substitution models for particular loci were determined using jModeltest v. 2.1.7 (Posada 2008) based on Bayesian information criterion (BIC) and were as follows: 1st analysis - K80+G (benA), K80+I (CaM), K80+G (act), K80+G (RPB2); 2nd analysis - K80+I (benA), K80+G (CaM), K80 (act), K80 (RPB2), HKY+I+G (tsr1), K80 (mcm7). In the first analysis, only unique sequences of four loci were used, i.e., benA, CaM, act and RPB2. The number of isolates of A. felis and A. pseudoviridinutans was reduced to two, because this clade was examined in detail in the second analysis based

Table 3   Partition-merging results and best substitution model for each partition according to Bayesian information criterion (BIC) as proposed by PartitionFinder v. 1.1.0. for combined dataset of benA, CaM, act and RPB2 genes. Dataset

Phylogenetic method

Partitioning scheme (substitution model)

Section Fumigati (Fig. 1)

Maximum likelihood

benA + CaM + act introns (TrNef+G); 3rd codon positions of benA (GTR+G); 1st codon positions of benA + CaM + act + RPB2 + 2nd codon positions of act + 3rd codon positions of act (TIM+I); 2nd codon positions of benA + CaM + RPB2 (HKY); 3rd codon positions of CaM + RPB2 (HKY+G)



Bayesian inference

benA + CaM + act introns (K80+G); 3rd codon positions of benA (GTR+G); 1st codon positions of benA + CaM + act + RPB2 + 2nd codon positions of act + 3rd codon positions of act (GTR+I); 2nd codon positions of benA + CaM + RPB2 (HKY); 3rd codon positions of CaM + RPB2 (HKY+G)

A. viridinutans clade (Fig. 5)

Maximum likelihood

benA + CaM + act introns (K80+G); 3rd codon positions of benA + CaM + RPB2 (TrN+G); 1st codon positions of benA + CaM + act + RPB2 + 3rd codon positions of act (TrN); 2nd codon positions of benA + CaM + act + RPB2 (F81)



Bayesian inference

benA + CaM + act introns (K80+G); 3rd codon positions of benA + CaM + RPB2 (HKY+G); 1st codon positions of benA + CaM + act + RPB2 + 3rd codon positions of act (HKY); 2nd codon positions of benA + CaM + act + RPB2 (F81)

Table 4   Overview of alignments characteristics used for phylogenetic analyses. Alignment characteristic

benA CaM act

Section Fumigati (Fig. 1) Length (bp) Variable position Parsimony informative sites

534 697 431 999 – – 268 322 234 280 – – 184 226 148 186 – –

RPB2 mcm7 tsr1

Combined dataset 2661 1104 744

A. viridinutans complex (Fig. 5) Length (bp) Variable position Parsimony informative sites

475 697 344 967 – – 115 168 102 135 – – 84 114 70 81 – –

2483 520 349

A. felis clade (Fig. 3) Length (bp) Variable position Parsimony informative sites

474 681 329 967 623 761 72 73 35 59 38 103 50 49 18 32 24 58

3835 380 231

150

on six loci. Three single-locus species delimitation methods, i.e., bGMYC (Reid & Carstens 2012), GMYC (Fujisawa & Barraclough 2013) and PTP (Zhang et al. 2013), and one multilocus species delimitation method STACEY (Jones 2017) were used to find putative species boundaries. The bGMYC and GMYC methods require ultrametric trees as an input, while PTP does not. Therefore, single locus ultrametric trees were constructed using a Bayesian approach in BEAST v. 2.4.5 (Bouckaert et al. 2014) with both Yule and coalescent tree models. We also looked at possible differences between strict and relaxed clock models, but since these parameters had no effect on the number of delimited species, only the results with strict clock model are presented here. Chain length for each tree was 1 × 10 7 generations with 25 % burn-in. The highest credibility tree was used for the GMYC method and 100 trees randomly sampled throughout the analysis were used for the bGMYC method. Both methods were performed in R v. 3.3.4 (R Core Team 2015) using bgmyc (Reid & Carstens 2012) and splits (SPecies’ LImits by Threshold Statistics) (Fujisawa & Barraclough 2013) packages. The nonultrametric trees for the PTP method were constructed using the ML approach in RAxML v. 7.7.1 (Stamatakis et al. 2008) and IQ-TREE v. 1.5.3 (Nguyen et al. 2015) with 1 000 bootstrap replicates. The PTP method was performed on the web server http://mptp.h-its.org/ (Kapli et al. 2017) with p-value set to 0.001. The multilocus species delimitation was performed in BEAST v. 2.4.5 with add-on STACEY v. 1.2.2 (Jones 2017). The chain length was set to 5 × 10 8 generations, priors were set as follows: the species tree prior was set to the Yule model, growth rate prior was set to lognormal distribution (M = 5, S = 2), clock rate priors for all loci were set to lognormal distribution (M = 0, S = 1), PopPriorScale prior was set to lognormal distribution (M = -7, S = 2) and relativeDeathRate prior was set to beta distribution (α = 1, β = 1 000). The output was processed with SpeciesDelimitationAnalyzer (Jones 2017). The species tree was inferred using *BEAST (Heled & Drummond 2010) implemented in BEAST v. 2.4.5. The isolates were assigned to a putative species according to the results of the above-mentioned species delimitation methods. The MCMC analysis ran for 1 × 10 8 generations, 25 % of trees were discarded as a burn-in. The strict molecular clock was chosen for all loci and population function was set as constant. Convergence was assessed by examining the likelihood plots in Tracer v. 1.6 (http://tree.bio.ed.ac.uk/software/tracer). We also constructed the phylogenetic tree based on concatenated alignment of all four loci in IQ-TREE v. 1.5.3 with 1 000 bootstrap replicates and the optimal partitioning scheme determined by PartitionFinder v. 2.1.1 (Lanfear et al. 2017). The validation of the species hypotheses was performed in BP&P v. 3.3 (Bayesian phylogenetics and phylogeography) (Yang & Rannala 2010). The isolates were assigned to the species based on the results of species delimitation methods and the species tree inferred with *BEAST was used as a guide tree. Three different combinations of the prior distributions of the parameters θ (ancestral population size) and τ0 (root age) were tested as proposed by Leaché & Fujita (2010), i.e., large ancestral population sizes and deep divergence: θ ~ G (1, 10) and τ0 ~ G (1, 10); small ancestral population sizes and shallow divergences among species: θ ~ G (2, 2000) and τ0 ~ G (2, 2000); large ancestral populations sizes and shallow divergences among species: θ ~ G (1, 10) and τ0 ~ G (2, 2000). The second analysis with six protein-coding loci, i.e., benA, CaM, act, RPB2, mcm7 and tsr1, consisted of the same steps as described above. Instead of PTP, we used the programme mPTP (Kapli et al. 2017) with IQ-TREE and RAxML trees as an input. Within the mPTP programme we used the following settings: Maximum likelihood species delimitation inference (option ML) and a different coalescent rate for each delimited

Persoonia – Volume 41, 2018

species (option multi). R package ggtree (Yu et al. 2017) and the programme densitree (Bouckaert 2010) were used for visualization of the phylogenetic trees. Mating experiments The MAT idiomorph was determined using the primer pairs alpha1 and alpha2 located in MAT1-1-1 locus (alpha box domain), and HMG1 and HMG2 primers located in MAT1-2-1 locus (high-mobility-group domain) as described by Sugui et al. (2010). The MAT idiomorphs were differentiated based on the different lengths of PCR products visualized by gel electro­ phoresis; absence of opposite MAT idiomorph was also verified in all isolates. The identity of PCR products was proved by DNA sequencing in several isolates (accession numbers in Table 1); product purification and sequencing were performed at Macrogen Europe (Amsterdam, The Netherlands) using terminal primers. Selected opposite mating type strains were paired within and between major phylogenetic clades on MEA and oatmeal agar (OA; Difco, La Ponte de Claix, France) plates and incubated at 25, 30 and 37 °C in the dark. The plates were sealed with Parafilm and examined weekly from the third wk of cultivation for two months under a stereomicroscope for the production of ascomata. The presence of ascospores was determined using light microscopy. Width and height of ascospores were recorded at least 35 times for each successful mating pair. Statistical analysis Statistical differences in the width and height of the ascospores of particular species and interspecific hybrids were tested with one-way ANOVA followed by Tukey’s HSD (honest significant difference) post hoc test in R v. 3.3.4 (R Core Team 2015). R package multcomp (Hothorn et al. 2008) was used for the calculation and package ggplot2 (Wickham 2009) for visualization of the results. Exometabolite analysis The extracts were prepared according to Houbraken et al. (2012). High-performance liquid chromatography with diodearray detection was performed according to Frisvad & Thrane (1987, 1993) as updated by Nielsen et al. (Nielsen et al. 2011). Fungi were incubated for 1 wk at 25 °C in darkness on CYA and yeast extract sucrose (YES) agars for exometabolite analysis. RESULTS Phylogenetic definition of AVSC In the phylogenetic analysis, 76 combined benA, CaM, act and RPB2 sequences were assessed for members of sect. Fumigati. The analysis was based on the modified alignment previously used by Hubka et al. (2017) and enriched by taxa from AVSC. In the Bayesian tree shown in Fig. 1, members of sect. Fumigati are resolved in several monophyletic clades. The analysis showed that AVSC is a phylogenetically well-defined group and the clade gained full support. Similarly, some other clades are well-supported by both BI and ML analyses including A. spinosus clade, A. brevipes clade, A. tatenoi clade, A. thermomutatus clade and A. fennelliae clade; A. spathulatus forms a singlespecies lineage distantly related to other clades. Other clades have moderate or low support and the species represented therein may differ based on genetic loci used for phylogenetic reconstruction and taxa included in the analysis. Heterothallic species are dispersed across sect. Fumigati (Fig. 1) but the majority of them cluster in AVSC and A. fumigatus clades. These two clades also encompass the highest number of human and animal pathogens in sect. Fumigati not only in terms of their number but also their clinical relevance.

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● A. aureolus NRRL 2244T ● „A. indohii“ IFM 53615T ■ A. acrensis IFM 57291T ● homothallic ■ A. acrensis CCF 4959 */* */* heterothallic/anamorphic: ● A. udagawae IFM 46972T -/● sexual state known ● A. udagawae IFM 54131 */* ■ sexual state unknown ● A. udagawae CCF 4475 */* ● A. udagawae CCF 4478 */99 ● A. wyomingensis CCF 4417T 0.97/89 benA + CaM + act + RPB2 */* ● A. wyomingensis CCF 5640 ● A. wyomingensis IMI 133982 ● A. siamensis IFM 59793T */* 0.98/71 -/● A. siamensis IFM 61157 ● A. felis CBS 130245T */* ● „A. pseudofelis“ NRRL 62903T */* ● „A. parafelis“ NRRL 62900T */* ● A. felis FRR 5679 */* 0.96/*/* ■ A. pseudoviridinutans NRRL 62904T ■ A. pseudoviridinutans IMI 182127 */* ■ A. pseudoviridinutans CBS 458.75 */96 ■ A. arcoverdensis IFM 61334T */* ■ A. arcoverdensis IFM 59922 ■ A. arcoverdensis CBS 121595 */* ■ A. frankstonensis CBS 142233T */99 ■ A. frankstonensis CBS 142234 */98 */99 ■ A. viridinutans NRRL 4365T ● A. fumigatus NRRL 163T */97 */* ■ A. oerlinghausensis CBS 139183T 0.99/70 ● A. fischeri NRRL 181T ■ A. fumisynnematus IFM 42277T */* A. fumigatus 0.99/71 ● A. lentulus NRRL 35552T ■ A. fumigatiaffinis IBT 12703T ■ A. novofumigatus IBT 16806T */99 */99 -/● A. paulistensis CBM-FA-0690T */98 */* ● A. takakii CBM-FA-884T -/A. spinosus clade ● A. laciniosus KACC 41657T */* ● A. coreanus KACC 41659T ● A. spinosus NRRL 5034T ● A. quadricinctus NRRL 2154T */* */85 -/● A. tsurutae CBM-FA-0933T A. brevipes clade */99 ■ A. duricaulis NRRL 4021T ■ A. brevipes NRRL 2439T -/T */97 ● A. tatenoi CBS 407.93 -/*/99 ● A. tatenoi NRRL 4584 A. tatenoi clade ● A. delicatus CBS 101754T T ● A. thermomutatus NRRL 20748 */* A. thermomutatus / ● Aspergillus sp. NRRL 1283 A. spathulatus clades ● A. spathulatus NRRL 20549T ■ A. marvanovae CCM 8003T */* -/● A. turcosus IBT 27921T -/■ A. unilateralis NRRL 577T -/● A. caatingaensis IFM 61335T ● A. multiplicatus IFM 53594T */99 A. unilateralis clade ● A. tsunodae IFM 57609T -/● A. nishimurae IFM 54133T */84 ● A. waksmanii NRRL 179T 0.95/*/83 ● A. assulatus IBT 27911T ■ A. tasmanicus CBS 283.66T 0.95/72 ● A. hiratsukae CBS 294.93T ■ A. brevistipitatus CCF 4149T */98 0.02 ■ A. conversis CCF 4190T -/- 0.95/● A. solicola NRRL 35723T 0.98/● A. australensis NRRL 2392T A. neoglaber clade -/*/75 ● A. papuensis CBS 841.96T -/T ● A. pernambucoensis IFM 61342 -/● A. galapagensis IBT 16756T -/● A. neoglaber NRRL 2163T ● A. shendaweii IFM 57611T -/● A. auratus NRRL 4378T */98 A. auratus clade ● A. stramenius NRRL 4652T ● A. denticulatus CBS 652.73T */* -/71 ● A. sublevisporus IFM 53598T A. fennelliae clade */95 ● A. huiyaniae IFM 57848T */99 ● A. fennelliae NRRL 5535T ● A. similanensis KUFA 0012T

Reproductive strategy

*/*

*/*

A. viridinutans clade

clade

A. clavatus NRRL 1T Fig. 1   Phylogenetic relationships of the sect. Fumigati members inferred from Bayesian analysis of the combined, 4-gene dataset of β-tubulin (benA), calmodulin (CaM), actin (act ) and RNA polymerase II second largest subunit (RPB2) genes. Bayesian posterior probabilities (PP) and Maximum likelihood bootstrap supports (BS) are appended to nodes; only PP ≥ 95 % and BS ≥ 70 % are shown; lower supports are indicated with a hyphen, whereas asterisks indicate full support (1.00 PP or 100 % BS); ex-type strains are designated by a superscript T; species names in quotes are considered synonyms; the bar indicates the number of substitutions per site. The tree is rooted with Aspergillus clavatus NRRL 1T. The reproductive mode of each species is designated by icons before the species name (see legend).

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Persoonia – Volume 41, 2018

RPB2

PTP RAxML PTP IQ-tree bGMYC coalescent bGMYC yule GMYC coalesent GMYC yule

CaM

PTP RAxML PTP IQ-tree bGMYC coalescent bGMYC yule GMYC coalesent GMYC yule

benA

PTP RAxML PTP IQ-tree bGMYC coalescent bGMYC yule GMYC coalesent GMYC yule PTP RAxML

act

PTP IQ-tree bGMYC coalescent bGMYC yule GMYC coalesent GMYC yule

100

100 88 83

90

100 99

100

74

A. udagawae clade 2 A. udagawae clade 3

94

100

0.98 0.99 1.00

1.00 1.00 1.00

0.84 0.88 1.00

100

100

A. aureolus

100 1.00 1.00 1.00

A. felis A. frankstonensis A. viridinutans A. arcoverdensis

100 1.00 1.00 1.00

A. wyomingensis A. pseudoviridinutans

100

100

A. acrensis

A. siamensis

100

100

100

A. udagawae clade 1

100

100

100 1.00 1.00 1.00

100

1.00 1.00 1.00

Fig. 2   Schematic representation of results of species delimitation methods in Aspergillus viridinutans species complex based on four genetic loci. The results of multilocus method (STACEY) are compared to results of single-locus methods (PTP, bGMYC, GMYC). The results of STACEY are shown as tree branches with different colours, while the results of single-locus methods are depicted with coloured bars highlighting congruence across methods. The displayed tree is derived from IQ-TREE analysis based on a concatenated dataset and is used solely for the comprehensive presentation of the results from different methods. The species validation analysis results (BP&P) are appended to nodes and shown in grey bordered boxes; the values represent posterior probabilities calculated in three scenarios having different prior distributions of parameters θ (ancestral population size) and τ0 (root age). The top value represents the results of analysis with large ancestral population sizes and deep divergence: θ ~ G (1, 10) and τ0 ~ G (1, 10); the middle value represents the results of analysis with large ancestral populations sizes and shallow divergences among species: θ ~ G (1, 10) and τ0 ~ G (2, 2000); and the bottom value small ancestral population sizes and shallow divergences among species: θ ~ G (2, 2000) and τ0 ~ G (2, 2000).

153

mPTP RAxML mPTP IQ-tree

bGMYC coalescent

bGMYC yule GMYC coalesent GMYC yule mPTP RAxML mPTP IQ-tree

bGMYC coalescent

bGMYC yule GMYC coalesent GMYC yule

mPTP RAxML mPTP IQ-tree

bGMYC coalescent

bGMYC yule GMYC coalesent GMYC yule

mPTP RAxML mPTP IQ-tree

bGMYC coalescent

bGMYC yule GMYC coalesent GMYC yule

mPTP RAxML mPTP IQ-tree

bGMYC coalescent

bGMYC yule GMYC coalesent GMYC yule mPTP RAxML mPTP IQ-tree

bGMYC coalescent

1.00 1.00 0.51

* ex-type of A. felis ** ex-type of A. parafelis *** ex-type of A. pseudofelis ▲ ex-type of A. pseudoviridinutans ▲▲ ex-type of A. fumigatus var. sclerotiorum

1.00 1.00 0.51

IFM 47021

CBS 458.75

IHEM 22515

▲▲

IFM 57289

IFM 55266

IMI 182127

82

73 100

CCF 5632

100

clade 3

100 1.00 1.00 1.00

100

1.00 1.00 1.00

82

100

A. aureolus (outgroup)

clade 2

83

▲ NRRL 62904

FRR 5679

CCF 5609

IFM 59564

CCF 5610

CCF 5614

FRR 5680

93

82

clade 1 0.99 0.99 0

CCF 4497

100

100

89

85

A. pseudoviridinutans

97

94

0.19 0.24 0

CCF 4002

**

NRRL 62900

NRRL 62901

IFM 60053

CCF 5624

CCF 5616

* 94

A. felis

75

CBS 130245

CCF 5628

CCF 5618

NRRL 62903

***

CCF 5622

CCF 4148

IFM 54303

CCF 5626

CBS 130249

CCF 5623

CCF 5617

bGMYC yule GMYC coalesent GMYC yule

CCF 5625

act

benA

CaM

mcm7

RPB2

tsr1

V. Hubka et al.: Species delimitation and hybridization in section Fumigati

Fig. 3   Schematic representation of results of species delimitation methods in Aspergillus felis clade based on six genetic loci. The results of multilocus method (STACEY) are compared to results of single-locus methods (mPTP, bGMYC, GMYC). The results of STACEY are shown as tree branches with different colours, while the results of single-locus methods are depicted with coloured bars highlighting congruence across methods. The displayed tree is derived from IQ-TREE analysis based on a concatenated dataset and is used solely for the comprehensive presentation of the results from different methods. The species validation analysis results (BP&P) are appended to nodes and shown in grey bordered boxes; the values represent posterior probabilities calculated in three scenarios having different prior distributions of parameters θ (ancestral population size) and τ0 (root age). The top value represents the results of analysis with large ancestral population sizes and deep divergence: θ ~ G (1, 10) and τ0 ~ G (1, 10); the middle value represents the results of analysis with large ancestral populations sizes and shallow divergences among species: θ ~ G (1, 10) and τ0 ~ G (2, 2000); and the bottom value small ancestral population sizes and shallow divergences among species: θ ~ G (2, 2000) and τ0 ~ G (2, 2000).

154

Species delimitation and validation in AVSC In the first analysis, four genetic loci were examined across species of AVSC, isolates of A. felis and its close relatives were reduced to two individuals, because a separate analysis based on six loci was performed for this clade. Eleven tentative species were delimited in AVSC using STACEY. The results are summarised in Fig. 2, the differences in the colour of the tree branches reflect species delimited by the analysis. The analysis supported recognition of three putative species in A. udagawae lineage, delimitation of A. acrensis (described below) from A. aureolus was not supported, other AVSC species were supported by STACEY without differences from their current concept. The results derived from STACEY were compared to those from three single-locus species delimitation methods. The consensual results from single-locus species delimitation methods are generally in agreement with the results of STACEY for the majority of species but vary greatly for A. udagawae, A. aureolus and A. acrensis lineages (Fig. 2). Recognition of three putative species in A. udagawae lineage was supported only based on the CaM locus, while based on benA locus, none of these three sublineages gained support. Various delimitation schemes were proposed by different single-locus species delimitation methods in the A. udagawae lineage based on the RPB2 gene (results even varied between the analyses based on different input trees for the PTP and GMYC methods), while five putative species were identically delimited based on the act locus. The methods relatively consistently supported delimitation of the A. acrensis lineage based on the RPB2 locus and similarly, bGMYC and GMYC methods supported this species based on the act locus. In contrast, lineages of A. acrensis and A. aureolus were not split by any method when analyzing benA and CaM loci. The species validation analysis results are appended to nodes of the tree in Fig. 2. A reasonable support is defined by posterior probabilities ≥ 0.95 under all three scenarios simulated by different prior distributions of parameters θ (ancestral population size) and τ0 (root age). Delimitation of all putative species (those delimited by STACEY, A. acrensis and A. aureolus) were supported by the posterior probability 0.98 or higher based on the analysis in BP&P v. 3.1 (Yang & Rannala 2010) under all three scenarios. The only exception was lower support for splitting of A. acrensis and A. aureolus; this scenario was supported by the posterior probabilities 0.84, 0.88, 1.00, respectively. Species delimitation and validation in A. felis clade and its relatives In the second analysis, six genetic loci were examined across isolates of A. felis, A. parafelis, A. pseudofelis and A. pseudo­ viridinutans. Only two tentative species, A. felis and A. pseudo­ viridinutans, were delimited in this clade using STACEY. The results are shown as branches designated by different colours in Fig. 3. The analysis did not support separation of A. pseudofelis and A. parafelis from A. felis; A. fumigatus var. sclerotiorum is included in the lineage of A. pseudoviridinutans. The results of three single-locus species delimitation methods were compared to those from STACEY, and the consensual results showed a general agreement (Fig. 3). Delimitation of A. pseudofelis from A. felis was not supported by any of the used methods. Only a negligible number of analyses supported delimitation of basal clades in A. felis as tentative species (designated as clade 2 and 3 in Fig. 3). But even in these minority scenarios, there were no clear consensual delimitation patterns that would support delimitation of A. parafelis. Interestingly, mPTP analysis based on act, benA, CaM (with RAxML trees as an input only), mcm7 and tsr1 loci together with GMYC analysis based on benA (only input tree based on coalescent tree model)

Persoonia – Volume 41, 2018

and act (only input tree based on Yule tree model) loci did not support delimitation of A. pseudoviridinutans from a robust clade of A. felis. An incomplete lineage sorting was observed between A. felis and A. pseudoviridinutans (Fig. 3) evidencing that there was probably an ancestral gene flow between these lineages. Two isolates from A. felis lineage (IFM 59564 and CCF 5610) have benA sequences that cluster with A. pseudoviridinutans while sequences of the remaining 5 loci placed them in the A. felis lineage (single-gene trees not shown). The species validation analysis results are appended to nodes of the tree in Fig. 3. Delimitation of A. felis and A. pseudo­ viridinutans gained absolute support in BP&P analysis (Yang & Rannala 2010) under all three scenarios simulated by different prior distributions of parameters θ (ancestral population size) and τ0 (root age). Delimitation of three putative species within A. felis lineage gained no support (posterior probability 0.51) under the scenario with small ancestral population sizes and shallow divergences among species: θ ~ G (2, 2000) and τ0 ~ G (2, 2000). Species tree The species tree topology was inferred with *BEAST (Heled & Drummond 2010) and is shown in Fig. 4. It was used as a guide tree during species validation using BP&P but it also represents the most probable evolutionary relationships between species in the AVSC. The analysis confirmed recombination between three subclades of A. felis (Fig. 4) which include also recently proposed species A. parafelis and A. pseudofelis thus representing the synonyms of A. felis. Similarly, the recombination between three subclades of A. udagawae rejected the hypothesis that they could be considered separate species (Fig. 4). clade 2

clade 2

A. udagawae

clade 3

clade 3

clade 1

clade 1

A. acrensis A. aureolus A. wyomingensis A. siamensis

clade 1

clade 2 clade 3

clade 1

A. felis

clade 2 clade 3

A. pseudoviridinutans

A. arcoverdensis A. frankstonensis A. viridinutans

A. lentulus (outgroup)

Fig. 4   Species tree inferred with *BEAST visualized by using DensiTree (Bouckaert 2010). All trees created in the analysis (except 25 % burn-in phase) are displayed on the left side. Trees with the most common topology are highlighted by blue, trees with the second most common topology by red, trees with the third most common topology by pale green and all other trees by dark green. On the right side, the consensus trees of the three most common topologies are displayed.

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Continent of origin ❶ Europe ❷ Asia ❸ Australia ❹ North America ❺ South America ❻ Africa

benA + CaM + act + RPB2

0.97/78

Reproductive mode H homothallic M1 heterothallic, MAT1-1-1 M2 heterothallic, MAT1-2-1 M heterothallic, MAT not determined Source of isolation animal

*/85 ❹ M2 CCF 4475

❹ M2 CCF 4494 ❹ M2 CCF 5636 ❹ M1 CCF 5637 0.98/80 ❹ M1 CCF 4476 -/0.01 ❹ M2 CCF 4491 ❹ M2 CMF ISB 1972 */88 clade 2 ❹ M2 CCF 4492 */89 ❸ M CCF 5639 */98 ❹ M2 CCF 4478 -/*/* ❹ M2 CCF 4481 ❷ M1 IFM 54131 clade 3 */* */98 ❷ M1 IFM 54745 ❷ M2 IFM 54132 -/❶ M2 CCF 5634 */* -/❹ M1 CCF 5638 ❹ M1 CMF ISB 2190 -/*/76 ❺ M1 IFM 46972T ❺ M2 IFM 46973 0.99/78 ❺ M1 IFM 62155 */79 ❷ M1 IFM 51744 clade 1 -/- ❷ M2 IFM 53868 ❷ M1 IFM 5058 -/❶/❷ M2 IFM 55207 ❹ M2 CCF 4479 ❶ M2 CCF 4960 */90 ❶ M1 CCF 4961 */92 ❶ M2 CCF 4959 */* ❺ M2 IFM 57290 ❺ M1 IFM 57291T ❺ H IFM 46584 */79 */* ❺ H IFM 53615 „A. indohii“ */* ❺ H IHEM 22515 ❻ H IFM 47021T ❹ M1 CCF 4413 ❹ M1 CCF 4415 */- ❹ M1 CCF 4420 ❹ M1 CCF 4414 ❹ M1 CCF 4412 ❹ M1 CCF 4417T -/❹ M2 CCF 4416 ❹ M2 CCF 4419 ❹ M2 CCF 4170 0.99/- ❹ M2 CCF 4418 ❹ M2 CMF ISB 1977 0.98/89 ❹ M1 CCF 4169 ❷ M2 IFM 59681 */* ❸ M2 CCF 5640 ❶ M1 IMI 133982 */* ❷ H IFM 59793T ❷ H IFM 61157 ❸ M1 CCF 5617 */❸ M1 CCF 5623 ❸ M1 CCF 5625 -/❸ M1 CCF 5627 -/- ❸ M2 CCF 5628 -/- -/❸ M1 CCF 5629 ❸ M2 CCF 5626 ❸ M2 CBS 130249 -/❸ M2 CCF 5622 */99 ❶ M1 CCF 4376 */68 ❶ M2 NRRL 62903 „A. pseudofelis“ ❹ M1 CCF 4148 ❸ M2 CCF 5619 */98 */95 ❸ M2 CBS 130245T ❸ M2 CCF 5618 -/❷ M1 IFM 54303 -/❸ M2 CCF 5609 */96 ❸ M1 CCF 5624 */clade 1 ❷ M1 IFM 60053 ❸ M CCF 5616 ❹ M2 CCF 4497 ❹ M2 CCF 4171 ❶ M CCF 4172 */71 ❶ M2 CCF 2937 ❶ M2 NRRL 62900 „A. parafelis“ ❶ M1 NRRL 62901 */99 ❹ M2 CCF 4498 -/❶ M2 CCF 4002 */* ❶ M2 CCF 4003 */70 ❸ M CCF 5614 clade 2 ❸ M2 FRR 5680 -/❸ M1 CCF 5610 */99 ❷ M2 IFM 59564 */90 ❸ M2 CCF 5611 ❸ M2 FRR 5679 clade 3 */99 ❷ M1 IFM 59502 -/❷ M1 IFM 59503 */* ❹ M1 NRRL 62904T */* ❷ M2 IMI 182127 */* ❹ M1 CCF 5632 */91 ❺ M2 IFM 57289 -/❷ M2 CBS 458.75 „A. fumigatus var. sclerotiorum“ ❷ M1 IFM 55266 ❺ M1 IFM 61339 */❺ M2 IFM 61362 -/❺ M1 IFM 61340 0.98/❺ M1 IFM 61334T ❺ M2 IFM 61338 */79 ❺ M2 IFM 61346 ❺ M2 IFM 61349 ❺ M2 IFM 61345 */76 ❺ M1 IFM 61337 ❺ M1 IFM 61333 ❷ M1 IFM 59922 */93 ❷ M1 IFM 59923 ❸ M1 CBS 121595 */85

-/-

A. udagawae

*/*

human soil/cave sediment

A. acrensis

other

A. aureolus

*/*

A. wyomingensis

-/-

A. siamensis

-/88

*/*

A. felis

A. pseudoviridinutans

A. arcoverdensis

*/* */92 */* */*

❸ M1 IFM

❸ M2 CBS 142233T ❸ M2 CBS 142234

47045T

A. frankstonensis A. viridinutans

A. lentulus NRRL 35552T

Fig. 5   Phylogenetic relationships of the Aspergillus viridinutans species complex members inferred from Bayesian analysis of the combined, 4-gene dataset of β-tubulin (benA), calmodulin (CaM), actin (act) and RNA polymerase II second largest subunit (RPB2) genes. Bayesian posterior probabilities (PP) and Maximum likelihood bootstrap supports (BS) are appended to nodes; only PP ≥ 90 % and BS ≥ 70 % are shown; lower supports are indicated with a hyphen, whereas asterisks indicate full support (1.00 PP or 100 % BS); ex-type strains are designated by a superscript T; species names in quotes are considered synonyms; the bar indicates the number of substitutions per site. The tree is rooted with Aspergillus lentulus NRRL 35552 T. The geographic origin and reproductive mode with MAT idiomorph (if known) is designated by icons before the isolate number while substrate of origin is designated by icons after isolate number (see legend).

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Persoonia – Volume 41, 2018

MAT 1-1-1

CCF 5628 CCF 5626 CCF 5622 CCF 5619 CBS 130245T CCF 5618 NRRL 62903T CCF 5609 CCF 4171 CCF 4498 NRRL 62900T FRR 5680 CCF 5611 FRR 5679

CCF 5625* CCF 5627 CCF 5617* CCF 5629

A. felis

CCF 4148 CCF 4376

= A. parafelis = A. pseudofelis

IFM 54303 IFM 60053 NRRL 62901 CCF 5610*

A. pseudoviridinutans

NRRL 62904T

IFM 59502 CCF 5632 IFM 55266

NO MATING

CMF ISB 2190

IFM 46972T IFM 51744 CCF 4476 CCF 5637 IFM 54131

IFM 57291T

A. acrensis

CCF 4961 CCF 4417T CCF 4413 CCF 4414 CCF 4169 IMI 133982 IFM 61339 IFM 61340 IFM 61334 IFM 61337 IFM 61333 IFM 59922 IFM 59923 CBS 121595

A. wyomingensis

Ascospore body width (μm)

A. arcoverdensis

a 6

b

NO MATING

c

CCF 4478 CCF 4492 IFM 54132 IFM 46973 IFM 55207 CCF 4479 CCF 4959 CCF 4960 IFM 57290 CCF 4419 CCF 4170 CCF 4416 CMF ISB 1977 IFM 59681

NO MATING IFM 61345 IFM 61338 IFM 61362

6

A. felis

A. wyomingensis

4

width height

3

IMI 182127

IFM 61349

A. udagawae 5

IFM 57289 CBS 458.75

IFM 61346

Ascospore body height (μm)

A. udagawae

MAT 1-2-1

a

b

c

5

4

3

2

Fig. 6   Schematic depiction of results of intraspecific mating experiments between opposite mating type isolates of heterothallic members of the Aspergillus viridinutans species complex. Only successful mating experiments are displayed by connecting lines between opposite mating type isolates; remaining mating experiments were negative. Isolates marked by asterisk were only crossed with ex-type strains of A. felis (CBS 130245 T), A. parafelis (NRRL 62900 T) and A. pseudofelis (NRRL 62903 T). Boxplot and violin graphs were created in R 3.3.4 (R Core Team 2015) with package ggplot2 (Wickham 2009) and show the differences between the width and height of ascospores of A. udagawae, A. wyomingensis and A. felis. Different letters above the plot indicate significant difference (P